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Exploring the var gene interactome with CRISPR/dCas9

ABSTRACT: Epigenetic regulation of mutually exclusive transcription within the var gene family is important for infection and pathogenesis of the malaria parasite Plasmodium falciparum. var genes are kept transcriptionally silent via heterochromatic clusters located at the nuclear periphery; however, only a few proteins have been shown to play a direct role in var gene transcriptional regulation. Importantly, the chromatin components that contribute to var gene nuclear organization remain unknown. Here, we adapted a CRISPR-based immunoprecipitation-mass spectrometry approach for de novo identification of factors associated with specific transcriptional regulatory sequences of var genes. Tagged, catalytically inactive Cas9 (“dCas9”) was targeted to var gene promoters or introns, cross-linked, and immunoprecipitated with all DNA, proteins, and RNA associated with the targeted locus. Chromatin immunoprecipitation followed by sequencing demonstrated that genome-wide dCas9 binding was specific and robust. Proteomics analysis of dCas9-immunoprecipitates identified specific proteins for each target region, including known and novel factors such as DNA binding proteins, chromatin remodelers, and structural proteins. We also demonstrate the ability to immunoprecipitate RNA that is closely associated to the targeted locus. Our CRISPR/dCas9 study establishes a new tool for targeted purification of specific genomic loci and advances understanding of virulence gene regulation in the human malaria parasite.

ORGANISM(S): Plasmodium falciparum

PROVIDER: GSE129397 | GEO | 2020/05/01

REPOSITORIES: GEO

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Exploring the virulence gene interactome with CRISPR/dCas9 in the human malaria parasite

Project description:The var multigene family encodes clonally variant surface antigens that are key to immune evasion and pathogenesis in the human malaria parasite, Plasmodium falciparum. Epigenetics and nuclear organization regulate the var gene family in a system of mutually exclusive expression; however, few factors have been shown to play a direct role in these processes. Thus, we adapted a CRISPR-based immunoprecipitation-mass spectrometry approach for identification of novel factors associated with var genes in their natural chromatin context. A tagged, catalytically inactive Cas9 (“dCas9”) was targeted to the promoters or introns of a subset of var genes and subjected to immunoprecipitation followed by label-free LC-MS/MS. A non-targeted dCas9 served as a control.

2020-07-15 | PXD013131 | Pride

P. falciparum Heterochromatin Protein 1 Marks Genomic Loci Linked to Phenotypic Variation of Exported Virulence Factors

Project description:Epigenetic processes are the main conductors of phenotypic variation in eukaryotes. The malaria parasite Plasmodium falciparum employs antigenic variation of the major surface antigen PfEMP1, encoded by 60 var genes, to evade acquired immune responses. PfEMP1 also mediates sequestration of infected erythrocytes in the microvasculature, which is directly linked to severe malaria outcomes. Antigenic variation of PfEMP1 occurs through in situ switches in mono-allelic var gene transcription, which is PfSIR2-dependent and associated with the presence of repressive H3K9me3 marks at silenced loci. Here, we show that the P. falciparum ortholog of heterochromatin protein 1 (PfHP1) binds to H3K9me3 and constitutes a major component of heterochromatin in perinuclear chromosome end clusters. High-resolution genome-wide chromatin immuno-precipitation demonstrates the striking association of PfHP1 with non-syntenic virulence gene arrays in subtelomeric and chromosome-internal islands. These include not only var genes but the majority of P. falciparum lineage-specific gene families coding for exported proteins involved in host-parasite interactions. Over-expression of PfHP1 resulted in decreased expression of a small number of (virulance) genes and indicated the presence of well-defined heterochromatic boundaries.. In summary, we uncover an unprecedented function of HP1 as a mayor regulator of virulence gene silencing and phenotypic variation, which will be instrumental for our understanding of this widely used survival strategy of unicellular pathogens. one experimental sample

2010-05-06 | E-GEOD-17029 | biostudies-arrayexpress

Epigenetic regulation of Plasmodium falciparum adaptive plasticity in the mosquito

Project description:P. falciparum undergoes antigenic variation during its life cycle in the human host. To accomplish this, the malaria parasite has developed mechanisms that ensure the expression of a single member of the var gene family to produce one of the PfEMP1 variant proteins. Here we carry out RNA-seq and ChIP-seq analyses of histone modifications to explore transcriptional and epigenomic changes taking place between the transmissible stages of the parasite i.e. gametocytes present in human blood and sporozoites present in the mosquito salivary glands. We find that most var genes are expressed at low levels in gametocytes but a single var gene is selected during the oocyst stage in the mosquito. Clonal expression of a specific var gene is accompanied by the establishment of specific histone modification patterns in the active and inactive genes. These modifications are epigenetically transmitted from the oocyst to the infective sporozoite stage, where expression of the active var gene is amplified by the transcription of an antisense lncRNA. The findings suggest a critical role for the mosquito immune system in determining the choice of var gene activation prior to transmission to the human host.

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Plasmodium falciparum Heterochromatin Protein 1 Marks Genomic Loci Linked to Phenotypic Variation of Exported Virulence Factors

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Expression data of gene knockout Plasmodium falciparum clones

Project description:The variant antigen, Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1), expressed on the surface of P. falciparum infected Red Blood Cells (iRBCs) is a critical virulence factor for malaria. Each parasite encodes 60 antigenically distinct var genes encoding PfEMP1s, but during infection the clonal parasite population expresses only one gene at a time before switching to the expression of a new variant antigen as an immune evasion mechanism to avoid the host’s antibody responses. The mechanism by which 59 of the 60 var genes are silenced remains largely unknown. We used microarrays to detail the global programme changes of gene expression caused by each gene knockout with a particular view towards which gene knockout results in transcriptional upregulation of the entire var gene family.

2013-05-25 | GSE47349 | GEO

PfSET2 is involved in genome organization of var gene family in Plasmodium falciparum [HiC]

Project description:The three-dimensional (3D) genome structure of human malaria parasite Plasmodium falciparum is highly organized and plays important roles in regulating coordinated expression patterns of specific genes such as virulence genes which are involved in antigenic variation and immune escape. However, the molecular mechanisms that control 3D genome of the parasite remain elusive. Here by analyzing genome organization in P. falciparum, we identify high-interacting regions (HIRs) with strong chromatin interactions telomeres and virulence genes loci. Specifically, HIRs are highly enriched with repressive histone marks (H3K36me3 and H3K9me3) and form the transcriptional repressive center. Deletion of PfSETvs, which controls H3K36me3 level, results in marked reduction of both intra-chromosomal and inter-chromosomal interactions for HIRs. Importantly, such chromatin reorganization coordinates with dymamic changes in epigenetic feature in HIRs and specifically activation of var genes. Our results uncover a fundamental mechanism that the epigenetic factor PfSETvs controls the 3D organization of heterochromatin in regulating the transcription activities of var genes family in P. falciparum.

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Evolutionarily selected overexpression of the cytokine BAFF enhances mucosal immune response against P. falciparum

Project description:A variant of the TNFSF13B gene (BAFF-var) increases the production of the cytokine BAFF, up-regulating humoral immunity and increasing the risk for certain autoimmune diseases. BAFF-var was evolutionarily advantageous, most likely by increasing resistance to malaria infection. We assessed experimentally the role of BAFF-var in response to malaria antigens. Lysates of erythrocytes infected with Plasmodium falciparum (iRBCs) or left uninfected (uRBCs, control) were used to treat PBMCs with distinct BAFF genotypes. PBMCs purified from BAFF-var donors and treated with iRBCs showed different levels of specific cells, immunoglobulins and cytokines as compared with BAFF-WT. In particularly a relevant differential effect on mucosal immunity B subpopulations have been observed. RNA sequencing of total B cells, purified from PBMC treated with iRBCs or uRBCs, identified several transcripts differentially expressed including some that encoded proteins critical for the response to malaria infection. These findings point to specific immune cells and molecules through which the evolutionary selected BAFF-var may have improved fitness during P. falciparum infection.

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